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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12N15/86—Viral vectors
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
  • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
  • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
  • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
  • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
  • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
  • C12N2810/851—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from growth factors; from growth regulators
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
  • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
  • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
  • C12N2810/859—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins

Definitions

• the present invention relates to a method for improving the efficiency of gene transfer into target cells in the fields of medicine, cell engineering, genetic engineering, developmental engineering, etc., and enabling efficient transformation of target cells, and related methods.
• a retroviral vector is a preferable means of gene transfer particularly for gene therapy in which long-term gene expression is desired, since it efficiently introduces a foreign gene of interest into cells and stably integrates it into its chromosomal DNA.
• the vector has been variously modified so as not to adversely affect the organism into which the gene has been introduced. For example, the replication function in the vector is deleted so that the vector itself used for gene transfer replicates in the cell and does not repeat unlimited infection (gene transfer).
• retrovirus-producing cells packaging cells
• the simplest method for highly efficient gene transfer into target cells is to co-culture the retrovirus-producing cells with the target cells.
• retrovirus-producing cells may be contaminated with the transfected target cells transplanted into the living body. There is.
• WO97Z188318 discloses that a functional substance other than fibrous cell nectin, such as fibroblast growth factor, improves gene transfer efficiency. Furthermore, the publication discloses that a similar improvement in gene transfer efficiency can be seen when a functional substance having retrovirus binding activity is mixed with another functional substance having cell binding activity. It is shown.
• Such a method for gene transfer using a functional substance enables highly efficient gene transfer without co-culturing retrovirus-producing cells and target cells. It is thought that the improvement of the gene transfer efficiency by this method is due to the fact that the functional substance co-locates the retrovirus and the target cell in close proximity, and the chance of interaction between the two increases.
• a main object of the present invention is to provide an improved method for improving gene transfer efficiency and efficiently transforming target cells in gene transfer into target cells by a retrovirus.
• FIG. 1 shows the structure of a high-mannose type sugar chain containing 9 mannose residues in the molecule.
• FIG. 2 is a graph showing the chemically modified CH-296 in Example 3 and the gene transfer efficiency (%) obtained thereby.
• FIG. 3 is a graph showing the relationship between the relative gene transfer efficiency (%) and the contact / binding time in the test of the removal effect of the virus infection inhibitor in Example 13;
• FIG. 4 is a graph showing the relationship between the relative gene transfer efficiency (%) in the test of the binding effect of the retrovirus and the functional substance using the centrifugal method in Example 15 and the virus binding operation.
• FIG. 5 is a graph showing the gene transfer efficiency (%) obtained by each of the centrifugation method and the centrifugal infection method in Example 15. Summary of the Invention
• the present inventors Before infecting a target cell, the present inventors contact a functional substance having a virus binding activity immobilized on a carrier with a retrovirus, and then subject the carrier to a washing operation. As a result, the gene transfer efficiency was found to be improved.
• target cells when retroviruses infect target cells, target cells can be identified by coexistence of target cell-specific antibodies, laminin, laminin-derived sugar chains, and high-mannose type sugar chains. And / or high-efficiency gene transfer.
• the gene transfer efficiency can be improved by subjecting the target cells to a suitable pretreatment before the gene transfer.
• a first aspect of the present invention is characterized in that a method for introducing a gene into a target cell by a retrovirus includes the following steps.
• step (1) is not particularly limited, but is performed, for example, for 1 hour or more, and preferably for 3 hours or more.
• the method is carried out by physically increasing the frequency of contact between the retrovirus and the retrovirus binding substance.
• the functional substance having a retrovirus binding activity used in the present invention is not particularly limited.
• fibronectin, fibroblast growth factor, type V collagen, polylysine, DEAE-dextran, a fragment thereof, or Substances having the same retrovirus binding activity can be used.
• a substance having a target cell binding activity is used as the above functional substance, or the above functional substance is used.
• the substance may be used in combination with another functional substance having a target cell binding activity.
• the functional substance having the target cell binding activity For example, cell-adhesive proteins, hormones, cytokines, antibodies, sugar chains, carbohydrates, metabolites, and the like can be used.
• retrovirus used for gene transfer in the present invention for example, a culture supernatant of a retrovirus-producing cell can be used.
• This culture supernatant may be obtained in the presence of a substance that promotes retrovirus production, for example, sodium butyrate.
• a second aspect of the present invention is characterized in that a retrovirus infects a target cell with a retrovirus in the presence of the following two types of functional substances in a method for introducing a gene into a target cell by a retrovirus.
• the target cell-specific antibody used in the present invention is not particularly limited, and examples include an antibody that recognizes a biological substance present on the surface of a target cell.
• a third embodiment of the present invention is characterized in that a retrovirus infects a target cell with a retrovirus in the presence of the following two types of functional substances in a method for introducing a gene into a target cell by a retrovirus.
• the functional substance having a retrovirus binding activity used in the second and third aspects of the present invention is not particularly limited. Examples thereof include fibronectin, fibroblast growth factor, type V collagen, polylysine, and DEAE. — Dextran, these fragments, or substances having the same retrovirus binding activity can be used.
• the functional substance may have a target cell binding activity. Further, the functional substance to be used may be used in a state of being immobilized on a suitable carrier.
• a fourth aspect of the present invention relates to a method for introducing a gene into a target cell using a retrovirus.
• the target cells Prior to the contact with the retrovirus, the target cells are cultured in a medium having a reduced Fe concentration.
• the medium used in the present invention is not particularly limited.
• a medium containing deferoxamine can be used, and the medium is preferably used in the presence of a functional substance.
• a fifth aspect of the present invention relates to a method for improving the retrovirus binding activity of a peptide or protein, characterized in that the peptide or protein is chemically modified.
• the chemical modification is not particularly limited, and examples thereof include activation of an amino acid residue of a peptide or protein and introduction of a basic residue.
• the method used for activating the amino acid residue is not particularly limited. For example, treatment with a water-soluble carbodiimide, and treatment with a water-soluble carbodiimide and a diamino compound are preferable.
• the chemically modified peptide or protein obtained by the method can be suitably used for retroviral gene transfer into target cells. Detailed description of the invention
• a recombinant retrovirus vector is usually used, and a replication-defective recombinant retrovirus vector is particularly preferable.
• the vector is replication-deficient so that it cannot replicate itself in infected cells and is non-pathogenic. These vectors can invade vertebrate cells, particularly host cells such as mammalian cells, and stably integrate the foreign gene inserted into the vector into their chromosomal DNA.
• a foreign gene to be introduced into a cell contains a recombinant retrovirus vector under the control of an appropriate promoter, for example, the promoter of the LTR present in the retrovirus vector or a foreign promoter. It can be inserted and used inside.
• an appropriate promoter for example, the promoter of the LTR present in the retrovirus vector or a foreign promoter. It can be inserted and used inside.
• a promoter and other regulatory elements cooperating with the transcription initiation site such as an enhancer sequence and a terminator sequence, may be present in the vector.
• the foreign gene to be introduced may be natural or artificially created, or may be a DNA obtained by linking DNA molecules of different origins by known means such as ligation. ,.
• any gene desired to be introduced into cells can be selected.
• exogenous genes encode enzymes and proteins associated with the disease to be treated, as well as intracellular antibodies (see, for example, W094 02610), growth factors, antisense nucleic acids, ribozymes, and proteins.
• Primers for example, refer to WO 9 OZl 3641 can be used.
• the retroviral vector used in the present invention may contain an appropriate marker gene that enables selection of the transfected cell.
• an appropriate marker gene that enables selection of the transfected cell.
• a drug resistance gene that confers resistance to an antibiotic on a cell a reporter gene that can identify a cell into which a gene has been introduced by an enzyme activity, and the like can be used as the gene.
• vectors examples include: MFG vector-1 (ATCC No. 68754), a- SGC vector-1 (ATCC No. 68755), LXSN vector radiotechnics (BioTechniques), Vol. 7, 980-9 90 (1 989)].
• PM5 neo vector [Exp. Hematol., Vol. 23, pp. 630-638 (1995)], and the like.
• the retrovirus vector contains the neomycin phosphotransferase gene as the primary gene. Therefore, cells transfected with the vector can be confirmed using the resistance to G418 as an indicator.
• these vectors are known packaging cell lines, for example, PG13 (ATCC CRL-10686), PG13 / LNc8 (ATCC CRL-10685), PA317 (ATCC CRL-9078), GP + E — 86 (ATCC CRL— 9642) or GP + env Am 1 2 (ATCC CRL—
• the vector can be prepared as packaged virus particles.
• known media for example, Dulbecco's modified Dudal's medium, Iscob's modified Dulbecco's medium, etc. can be used. Is commercially available from, for example, Gibco.
• Various components can be added to these media depending on the type of cells targeted for gene transfer and other purposes. For example, serum or various cytokines can be added for the purpose of promoting or inhibiting the growth and differentiation of target cells.
• calf serum for example, calf serum (CS), fetal calf serum (FCS) and the like can be used.
• cytokines include interleukins (IL-3, IL-6, etc.), colony stimulating factors (G-CSF, GM-CSF, etc.), stem cell factors (S
• cytokines those having an action according to the purpose may be selected, and if necessary, they may be used in combination.
• a sample containing a retrovirus for example, a culture supernatant of a virus-producing cell
• the preparation method is not particularly limited.
• the present invention is characterized in that a retrovirus is infected to a target cell in the presence of a functional substance having a binding site for a reticulovirus.
• a retrovirus is infected to a target cell in the presence of a functional substance having a binding site for a reticulovirus.
• the term “effective amount” refers to gene transfer into a target cell by a retrovirus. Is an amount effective to cause transformation of the target cell, and an appropriate amount is selected depending on the functional substance used and the type of the target cell. This amount can be determined, for example, by measuring the gene transfer efficiency by the method described herein.
• target cell binding activity as used herein includes not only an activity of substantially binding to cells but also an activity having an activity of maintaining contact with target cells in a solution. This activity can also be measured from its contribution to gene transfer efficiency as described above. Furthermore, the gene transfer efficiency means the transformation efficiency.
• the above-mentioned functional substance can be used in a state of being dissolved in a solution or a state of being fixed on a suitable carrier.
• the carrier for immobilizing the functional substance is not particularly limited, but usually a cell culture container or a bead-like carrier is used.
• a liquid sample containing a retrovirus for example, a virus supernatant
• a carrier on which a functional substance having retrovirus binding activity is immobilized After washing the carrier, gene transfer is carried out with high efficiency by contacting the carrier with the target cells as they are or by adding virus particles eluted from the carrier by an appropriate method to the target cells.
• the functional substance having a retrovirus binding activity used herein may be one having a target cell binding activity, and a functional substance having a retrovirus binding activity and a functional substance having a target cell binding activity May be used in combination.
• the step of bringing the liquid sample containing the retrovirus into contact with the carrier on which the functional substance having the retrovirus binding activity is immobilized is not particularly limited, but may be, for example, 1 hour or more, preferably 3 hours or more. Will be implemented.
• the conditions such as temperature are not particularly limited.
• the reaction can be performed at room temperature or at 37 ° C.
• a low temperature of about 4 ° C may be used depending on the stability of the virus.
• the carrier for immobilizing the functional substance may be appropriately selected according to the purpose.
• the gene transfer step can be started only by adding target cells.
• the carrier for example, phosphate buffered saline or Hank's saline can be used, as well as culture of target cells.
• a liquid medium to be used can also be used.
• the retrovirus can be more efficiently bound to the functional substance.
• Such physical means are not particularly limited, and for example, shaking, filtration or centrifugal force can be used.
• the method using centrifugal force is as follows. Specifically, a liquid sample containing a retrovirus is added to a centrifuge tube in which a functional substance having a retrovirus binding activity is immobilized on the bottom, and then the centrifuge tube is used. For centrifugation.
• the retrovirus sediments at the bottom of the centrifuge tube due to centrifugal force, so that the frequency of contact between the retrovirus and a functional substance having a retrovirus binding activity increases, and the binding efficiency increases.
• a higher gene transfer efficiency can be obtained without imparting physical stress to the cell unlike the method of infecting a virus by sedimenting the virus on the cell by centrifugal force (WO95 / 10619). Is the way.
• the substance removed by the method of the present invention includes, for example, a retrovirus infection inhibitor derived from packaging cells contained in the virus supernatant [Human Gene Therapy, Vol. 8, No. 1459- 1467 (1997), ⁇ Journal of Obir's (J. Virol.) Vol. 70, pp. 6468-6473 (1996)]
• Substances added for the purpose of promoting production for example, phorbol 12-myristate 13-acetate (TPA) ⁇ dexamethasone [Gene Therapy, Vol. 2, pp. 547-551 (195) And sodium butyrate described above.
• the functional substance having a retrovirus binding activity used in the present invention is not particularly limited, and examples thereof include a heparin-II binding region of fibronectin, fibroblast growth factor, type V collagen, polylysine, DEAE-dextran and the like. Also, a substance functionally equivalent to these functional substances, for example, a functional substance having a heparin binding site can be used. A mixture of the functional substances; A polypeptide containing a functional substance, a polymer of the functional substance, a derivative of the functional substance, and the like can be used.
• chemical modification methods include a method of activating an amino acid residue on a functional substance to be used and a method of introducing a basic residue into the substance.
• a method of activating an amino acid residue on a functional substance to be used and a method of introducing a basic residue into the substance.
• a free carboxyl group in a peptide or a functional substance consisting of a protein with a water-soluble carbodiimide for example, 1-ethyl-13-dimethylaminopropylcarbodiimide hydrochloride
• the retrovirus binding activity can be enhanced by introducing a basic residue, for example, an amino group, onto a functional substance by utilizing the carboxyl group thus activated.
• the functional substance having a target cell binding activity used in the present invention is not particularly limited.
• a functional substance having a ligand that binds to a target cell is used.
• the ligand include cell adhesive proteins, Examples include hormones and site proteins, antibodies against cell surface antigens, polysaccharides, glycoproteins, glycolipids, sugar chains derived from glycoproteins and glycolipids, and metabolites of target cells.
• a polypeptide containing the functional substance, a polymer of the functional substance, a derivative of the functional substance, a functional equivalent of the functional substance, and the like can also be used.
• An antibody that specifically binds to a target cell is particularly useful for introducing a gene into a specific cell with high efficiency.
• the antibody that can be used in the present invention is not particularly limited, and an antibody against an antigen expressed in a target cell into which a gene is to be introduced can be appropriately selected and used.
• Such antibodies can be produced by known methods. Many antibodies are currently commercially available, and these can also be used. These antibodies may be either polyclonal antibodies or monoclonal antibodies as long as they have desired properties such as cell specificity.
• an antibody or an antibody derivative modified by a known technique for example, a humanized antibody, a Fab fragment, a single-chain antibody, or the like can also be used.
• Leukocyte antigens known as CD antigens
• CD antigens have been examined in detail for their expression in various cells. Therefore, C expressed in the target cells of interest
• the gene can be transferred to the target cell with high specificity. For example, gene transfer can be directed to helper T cells when an anti-CD4 antibody is used, and to hematopoietic stem cells when an anti-CD34 antibody is used.
• laminin which is a glycoprotein
• the gene can be efficiently introduced into various target cells, for example, blood cells.
• Laminin which can be used in the present invention may be derived from a mouse or human, or may be a fragment thereof as long as it has a binding activity to a target cell.
• the sugar chain plays an important role in gene transfer using laminin. Therefore, sugar chains cut out by a method known from laminin can also be used in the method of the present invention.
• glycoproteins having a high mannose type N-linked sugar chain similar to laminin, sugar chains cut out therefrom, and chemically synthesized sugar chains can also be used in the present invention.
• those in which the above sugar chains are bound to a substance such as a protein can also be used.
• those in which a sugar chain is bound to a functional substance having a retrovirus binding activity can be suitably used for gene transfer.
• the above-mentioned high-mannose type sugar chain is not particularly limited as long as it has 1 to 20 residues of mannose in the molecule, but those having a mannose residue at the non-reducing end thereof are the present invention. Preferred for the method of the invention.
• the sugar chain is combined with other appropriate molecules such as monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, proteins, lipids, and other artificial molecules such as synthetic polymers. Can also be used.
• Typical high-mannose type sugar chains derived from living organisms include those having the structure of (Mannose) n — (GlucNAc) 2 [Protein, nucleic acid, enzyme, Vol. 43, Vol. 2631 ⁇ 2639 (1992)].
• Functional substances such as those described above can be obtained from naturally occurring substances and artificially produced (for example, produced by recombinant DNA technology or chemical synthesis technology). In addition, it can be produced by a combination of a naturally occurring substance and an artificially produced substance.
• a functional substance having a retrovirus binding site and a functional substance having a target cell binding site may be used. Or a functional substance having a retrovirus binding site and a target cell binding site on the same molecule can be used.
• these functional substances those which do not substantially contain other proteins which coexist in nature are used.
• a combination of these functional substances or functional substances can be combined with a medium used for culturing target cells, a cell growth factor, and the like to form a kit for gene transfer.
• fibronectin and fragments thereof used in the method of the present invention are described, for example, in Journal “OB. Biol. Chem.”, Vol. 256, Page 7277 (1981), Journal “OB”. Cell'Biology, J. Cell. Biol., Vol. 102, p. 449 (1986), Journal of Cell Biology, Vol. 105, p. 489 (1987) Can be produced in substantially pure form from materials of natural origin. Also, the recombinant DNA technology can be used for production according to the method described in US Pat. No. 5,198,423.
• a fibronectin fragment containing the heparin-1 II region which is a retrovirus binding site, such as CH-296 and ⁇ -271, ⁇ -296, C ⁇ -271 used in the following examples.
• Recombinant polypeptides and methods for obtaining them are described in detail in this patent. As described in the above gazette, these fragments were reported to FERM P-10721 ( ⁇ —296) by the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry at 1-3 1-3 Higashi, Tsukuba, Ibaraki Prefecture.
• H-296 represents a polypeptide binding region to VLA-4
• CH-271 represents a peptide binding region to VLA-5
• CH-296 represents both.
• Retroviral infection can be performed by conventional methods, for example, by incubation at 37 ° C and 5% carbon dioxide. These conditions and incubation time may be appropriately changed depending on the target cell and the purpose.
• G is the target cell. If the cells are in the early stage, they will not be infected by the retrovirus, so it is preferable that the cells be induced into the cell cycle by a preliminary stimulus.
• the target cells should be treated with an appropriate target cell growth factor prior to retroviral infection.
• Culture in the presence of for example, in the case of gene transfer into bone marrow cells or hematopoietic stem cells, various site strengths such as interleukin-13, interleukin-6, and stem cell factor are used for preliminary stimulation.
• Activation of the transferrin receptor is not particularly limited, but may be performed in a medium with a limited Fe concentration.
• the treatment can be carried out by treating the target cells.
• a medium in which Fe in the medium is made clear with the addition of deferoxamine can be used.
• gene transfer by transferrin activation is also performed in the presence of the above-mentioned functional substance.
• Cells targeted for gene transfer by the method of the present invention are not particularly limited.
• stem cells hematopoietic cells, non-adhesive low-density mononuclear cells, adherent cells, bone marrow cells, hematopoietic stem cells, peripheral blood Stem cells, umbilical cord blood cells, fetal hematopoietic stem cells, embryogenic stem cells, embryonic cells, primordial 'jam' cells (primordial germ cel l), oocytes, oocytes, ova, spermatocytes, sperm , CD34 + cells, C-kit + cells, pluripotent hematopoietic progenitor cells, monopotent hematopoietic progenitor cells, erythroid progenitor cells, lymphocyte mother cells, mature blood cells, lymphocytes, B cells, T cells, Use of fibroblasts, neuroblasts, neural cells, endothelial cells, vascular endothelial cells, hepatocyte
• Hematopoietic cells obtained from blood or bone marrow are relatively easy to obtain, and techniques for culturing and maintaining them have been established. Therefore, they are suitable for using the method of the present invention.
• hematopoietic stem cells, CD34 positive cells, C-kit positive cells, pluripotent hematopoietic progenitor cells, and other blood-based progenitor cells are suitable as target cells. ing.
• gene therapy using hematopoietic stem cells as target cells can be performed by the following operation.
• hematopoietic stem cells such as bone marrow tissue, peripheral blood, and umbilical cord blood
• a mononuclear cell fraction containing hematopoietic stem cells is prepared by a method such as density gradient centrifugation, or CD34 and / or CD34.
• purify hematopoietic stem cells using C-kit and one molecule of cell surface marker.
• the material containing these hematopoietic stem cells is, if necessary, subjected to preliminary stimulation using an appropriate cell growth factor or the like, and then the recombinant retrovirus vector into which the target gene is inserted by the method of the present invention. Infect.
• the transfected cells thus obtained can be transplanted into a recipient, for example, by intravenous administration.
• Recipe En The donor is preferably Donna itself, but it is also possible to perform allogeneic transplantation. For example, when umbilical cord blood is used as a material, allogeneic transplantation is performed.
• Gene therapy that targets hematopoietic stem cells complements genes that are missing or abnormal in patients. For example, gene therapy for ADA deficiency and Gaucher's disease.
• a drug resistance gene may be introduced into hematopoietic stem cells, for example, to alleviate the damage of hematopoietic cells caused by chemotherapeutic agents used for treating cancer or leukemia.
• tumor vaccine therapy has been studied in which a gene for cytokines is introduced into cancer cells and then their growth ability is removed and returned to the patient's body to enhance tumor immunity [ Human 'Gene' Therapy, Volume 5, Volume 1 53
• Human immunodeficiency virus is thought to introduce a gene that encodes a nucleic acid molecule (antisense nucleic acid, ribozyme, etc.) that interferes with HIV replication and gene expression into T cells infected with (human immunodeficiency virus).
• a nucleic acid molecule antisense nucleic acid, ribozyme, etc.
• the use of the present invention makes it possible to carry out gene transfer with high efficiency and high specificity for target cells.
• the method of the present invention does not require special equipment and devices, and is effective for various retrovirus vectors and target cells.
• H-271 a polypeptide derived from human fibronectin
• a polypeptide derived from human fibronectin is a recombinant plasmid containing DNA encoding the polypeptide, Escherichia coli containing pHD101, Escherichia coli HB O1 / pHDI O (FERM BP — 2264) And prepared by the method described in US Pat. No. 5,198,423.
• H-296 a polypeptide derived from human fibronectin
• FERMP — 10721 a polypeptide derived from human fibronectin
• Polypeptide CH-271 was prepared by the following method.
• E. coli and Escherichia coli HBlOl / pCHlOl were used and cultured according to the method described in the above publication, and CH-271 was obtained from the culture.
• Polypeptide CH-296 was prepared by the following method.
• E. coli Escherichia coli HB101 / pCH102 (FERMBP-2800) was used and cultured by the method described in the above-mentioned publication, and CH-296 was obtained from the culture.
• FERMBP-2800 Escherichia coli HB101 / pCH102
• Polypeptide C-274 was prepared by the following method.
• Co1V a polypeptide derived from type V collagen and having retrovirus binding activity, was prepared according to the method described in International Publication WO 97/18318.
• Example 2
• GP + E-86 introduced with a retroviral plasmid containing the neomycin phosphotransferase gene, PM5 neo vector-1
• Cells (ATCC CRL—9642) are Dulbecco's modified Eagle containing 10% fetal calf serum (FCS, Gibco) and 50 units / ml penicillin and 50 g / ml streptomycin (both from Gibco). Medium (DMEM, Bio-Ittsutaka) ).
• FCS 10% fetal calf serum
• FCS fetal calf serum
• DMEM Bio-Ittsutaka
• the supernatant containing PM5 neovirus was prepared by adding 4 ml of DMEM containing 10% FCS to a plate (1 Ocm diameter gelatin-coated cell culture dish, manufactured by Iwaki Glass Co., Ltd.) on which the above producing cells were grown in semi-confluent. It was prepared by culturing and culturing after cultivation. The collected culture supernatant was filtered through a 0.45 micron filter (Millipore) to obtain a virus supernatant stock, which was stored at 180 ° C until use.
• a retroviral plasmid containing a neomycin phosphotransferase gene and an Enhanced Green Fluorescent Protein (EGFP) gene an ecotopic packaging cell transfected with pLEIN (Clontech).
• BOSC23 Proceedings of the 'National' Academy 'of the Sciences of the US A, Vol. 90, pp. 8392-8396 (1993)]
• One zing cell ⁇ CRIP [Proceedings of the National Academy of Sciences, Inc., Science of Ob USA, Vol. 85, No. 6460-646
• a virus supernatant was prepared by the same operation as above.
• a virus prepared from BOS C23 cells is referred to as Eco-EGF ⁇
• a virus prepared from ⁇ CRIP cells is referred to as Ampho-EGFP.
• retrovirus plasmids containing the neomycin phosphotransferase gene, TKNeo Vector-1 [Jana-Ob'Experimental-Medicine (J. Exp. Med.), Vol. 178, pp. 529-536. , (1993)]
• a virus supernatant was prepared from GP + EnV Am12 cells (ATCC CRL—9641) in the same manner as described above.
• the DMEM used contained 10% litter serum (CS, manufactured by Gibco) instead of FCS.
• the titer of the above-mentioned virus supernatant was NIH 3T3 cells (ATCC CRL-1
• H-271, H-296, C-274, CH-271, CH-296, Co IV human basic fibroblast growth factor (b FGF, manufactured porogen Co.), Te defector (Tena SC i n, Gibco), epidermal growth factor (EGF, Takara Shuzo Co., Ltd.) or 2. /. ⁇ Add 50 ⁇ l per well of serum serum albumin (BSA, Sigma), and allow to stand at 4 ° C. Then, place the plate in phosphate buffered saline (PBS, Roman Industries). The washing operation was performed twice.
• BSA serum serum albumin
• PBS phosphate buffered saline
• Mouse leukemia cells L1210 grown in RPMI 1640 medium (manufactured by Bio-Wita Corporation) supplemented with 10% FCS, 50 units ml of penicillin and 50 ⁇ g Zml of streptomycin. (ATCC CC L- 2 1 9) 1 0 4 pieces and, the PM 5 neo virus supernatant (1 0 4 cfu / ml) 5 0 ⁇ 1 was ⁇ Ka ⁇ above microplate 1 Uweru. After culturing this for 24 hours, the medium was exchanged for one containing G4 18 (manufactured by Gibco) at a final concentration of 0.75 mg / ml, and culturing was continued for another 48 hours.
• G4 18 manufactured by Gibco
• the assay for G4 18 -resistant cells was carried out by partially modifying the method of S. Kim et al. [Gene 'Therapy, Vol. 3, pp. 108-120 (pp. 1996)], and using Premix WST- The color development of one reagent (Takara Shuzo) was performed by measuring the absorbance at 45 Onm. After culturing, add 10 ⁇ l of WST-1 reagent per 100/1 culture solution, and incubate at 37 ° C for 4 hours.Check the absorbance at 450 nm and 65 Onm with a microplate reader. The difference was measured and the difference (45 Onm-65 Onm) was calculated. The value obtained in the 2% BSA-coated, carbodiimide-untreated group was used as the background. 3 times Table 1 summarizes the results of these tests. table 1
• NH 2 manufactured by Nakarai] or trimethylene 1 OmgZm 1 1-ethyl-3-dimethyl containing various concentrations of diamine [NH 2 (CH 2 ) 3 NH 2 , manufactured by Nacalai] or putrescine [NH 2 (CH 2 ) 4 NH 2 , manufactured by Nacalai]
• a solution of aminopropylcarbodiimide hydrochloride (manufactured by Sigma) in PBS (pH 5.8) was added in an amount of 65 ⁇ l each, and the mixture was incubated at 37 ° C. for 2 hours. By this operation, an amino group was introduced into the carboxyl group on the molecule of CH-296 via carbodiimide. After washing the plate three times with PBS, the plate was sequentially blocked with 2% glycine ZPBS and 2% B SAZPBS.
• GP + E86 cells transfected with the retroviral vector plasmid pLEIN were cultured in DMEM containing 10% CS, and the supernatant was collected.
• a virus solution diluted to 1 ⁇ 10 5 cf xi / 1 with this supernatant is added in 0.5 ml per 1 ⁇ l of the above plate, incubated for 4 hours, and further 1 ⁇ 10 5 Four NIH / 3T3 cells were added and cultured for 2 days.
• the cells After completion of the culture, the cells are collected and washed with a buffer for cell detachment (manufactured by Bio-Wita Corporation), and then subjected to flow cytometry using FACS vantage (manufactured by Becton Dickinson) (excitation wavelength: 488 nm).
• FACS vantage manufactured by Becton Dickinson
• the EGFP-expressing cells were analyzed based on the fluorescence wavelength of 515 to 545 nm), and the virus binding ability to the plate was represented by the efficiency of gene transfer into the cells.
• Figure 2 shows the results.
• the virus binding ability increased as the concentration of the diamino compound used in the amino group introduction reaction increased.
• putrescine, trimethylenediamine, and ethylenediamine were used in the reaction at 2 mM, the virus binding ability was about twice as high as that of untreated CH-296.
• Gene transfer was performed using a combination of mouse laminin (manufactured by Gibco) or hytraminin (manufactured by Takara Shuzo) and a functional substance having a virus binding activity.
• the 24-well microplate for uncultured cell culture (Falcon) used in the experiment was coated with these functional substances by the following two methods.
• Precoat method A solution of a functional substance having a virus binding activity is added to a plate, allowed to stand at 4 ° C. at room temperature, and then the solution is removed. Then add laminin solution and incubate at 37 ° C for 2 hours. After blocking with 2% BSA, wash the plate with PBS.
• the functional substance solution used for the coating operation was added at 0.5 ml / well.
• the plate was coated by the cocktail method.
• the gene transfer efficiency is shown in%. Table 4
• the plate was coated by the cocktail method.
• the gene transfer efficiency is shown in%.
• the plate was coated by the cocktail method.
• the transduction efficiency is shown in%.
• Bone marrow cells were collected from the femur of a C3H / He female mouse (Japan SLC), and the obtained cells were subjected to density gradient centrifugation using Phyco-lupac (1.0875 g / ml, Pharmacia). A low density mononuclear cell fraction was prepared. Obtained After washing the cells with PBS, Ery- Lysis buffer (1 55 mM NH 4 C 1 , 10 mM KH C0 3, 0. ImM EDTA, p H 7. 4) dissolved erythrocytes, further washed with PBS .
• bone marrow cells to anti-mouse CD 1 1 7 antibody manufactured by Faminge emissions Co., Ltd.
• the cells were washed with PBS containing 5 mM EDTA and 0.5% BSA, and then suspended in the same buffer.
• microbeads conjugated secondary antibody Miltenyi Biotec
• 20 // 1 added per 10 7 cells allowed to react for 30 minutes at 4 ° C, resuspended and washed in the above buffer.
• Cells bound to the microbeads were collected using a MACS system (Mirtu Biotech) to obtain C-kit positive cells.
• mouse C-kit positive bone marrow cells Prior to virus infection, mouse C-kit positive bone marrow cells were primed based on the method of Luskey et al. [Blood, Vol. 80, pp. 396-402 (1992)]. . That is, 20% FCS, 20 ng Zml recombinant mouse interleukin-13 (Genzam), 5 OngZml recombinant human interleukin-6 (Genzam), 10 OngZml recombinant mouse stem cell factor (Jenzym) The cells were cultured at 37 ° C. for 2 days in a Hi-MEM (manufactured by Bio-Wita Corporation) containing 50 units of Zml of penicillin and 50 igZml of streptomycin in the presence of 5% carbon dioxide.
• Hi-MEM manufactured by Bio-Wita Corporation
• a 24-well microplate for untreated surface cell culture was coated by a cocktail method using mixtures of mouse laminin at various concentrations and 80 igZml of CH-271. / o Blocked with BSA for 30 minutes, and further washed with PBS. As a control, a plate using 2% BSA instead of CH-271 was also prepared.
• the gene transfer efficiency was shown in%. As shown in Tables 6 and 7, C-kit-positive bone marrow cells were retrovirus-infected on a plate on which mouse laminin and a functional substance having virus binding activity, CH-271, were coated by the cocktail method. In this case, it was clarified that a very high gene transfer efficiency enhancement effect was observed. The gene transfer efficiency when combined with Ramien was increased by a maximum of 5.7 times compared to when CH-271 alone was used.
• the gene transfer efficiency was shown in%.
• mice were collected from spleens of 6-8 week old C 3 H / He female mice and passed through a 100 // m mesh (Falcon) to remove residues. The obtained cells were washed with Hanks' balanced salt solution containing 10% FCS (HBS S, manufactured by Bio-Wita Corporation), and erythrocytes were lysed with an Ery-Lysis buffer, followed by washing with HBSS. The resulting cells were passed through a 30 ⁇ m mesh (Milteni Biotech) to remove residues, and then CD3 positive.
• HBS S Hanks' balanced salt solution containing 10% FCS
• erythrocytes were lysed with an Ery-Lysis buffer, followed by washing with HBSS.
• the resulting cells were passed through a 30 ⁇ m mesh (Milteni Biotech) to remove residues, and then CD3 positive.
• T cell concentration column manufactured by RL & D Systems.
• Mouse CD3-positive T cells used in virus infection experiments were 10% FCS, 50 units on a petri dish on which anti-mouse CD3 and CD28 antibodies (both l / g / ml, Pharmingen) were immobilized.
• Pre-stimulation was performed by culturing in RPMI 1640 medium (manufactured by Bio-Wita Corporation) containing Zml of penicillin and 50 gZml of streptomycin at 37 ° C for 2 days in the presence of 5% carbon dioxide.
• a 24-well microplate was coated in the same manner as in Example 5.
• the microplate over preparative to 10 5 per Ueru CD 3 positive T cells with 0.51111 of £ co-EGF P virus supernatant (10 5 cfu / ml) was added to perform a viral infection of 3 hours was.
• the gene transfer efficiency was shown in%. As shown in Table 9, it was revealed that the efficiency of gene transfer to mouse CD3-positive T cells was increased by the presence of lamin.
• a 96-well microplate was prepared in the same manner as in Example 5 using a mixture of 50 // 1 of 5 ⁇ g Zml of mouse laminin per well and 80 / ⁇ 1 After that, the plate was treated with various enzymes having sugar chain-cleaving activity, and the effect on gene transfer efficiency was examined.
• Endo- ⁇ -N-acetylglucosaminidase H manufactured by Seikagaku Corporation
• Endo- (3-galactosidase Endo-) 3_galactosidase, manufactured by Seikagaku Corporation
• ⁇ -mannosidase ⁇ -Mannosidase, manufactured by Seikagaku Corporation Co., Ltd.
• Peptide F (Peptide: N-glycosidase F, manufactured by Takara Shuzo Co., Ltd.) was prepared with 250 mM U / ml enzyme solution using 100 mM Tris-HCl buffer (pH 8.6). 5 0 ⁇ dispensed at 1 minute, 3 The reaction was performed at 7 ° C for 20 hours. Thereafter, the cells were washed three times with PBS, and then used for a virus infection experiment.
• soybean agglutinin prepared from defatted soybean powder (manufactured by Sigma) using Sepharose CL-2B (manufactured by Pharmacia) to which lactose is immobilized, contains 10 mM calcium chloride after heat denaturation
• the digestion was carried out using 20 mg of actinase E (manufactured by Kaken Pharmaceutical Co., Ltd.) in 20 ml of 50 mM Tris-HCl buffer (pH 7.2) at 37 ° C. for 2 days and nights.
• a microplate was prepared in which CH-271 and (Mannose) 9 — (GlucNAc) 2 —Asn were covalently immobilized. That is, a 96-well carboplate (manufactured by Sumitomo Berkit) was activated with a 4 mg Zm1 aqueous carbodiimide solution at 37 ° C for 2 hours, and then washed three times with sterile water.
• a 24-well microplate for untreated cell culture was coated according to the method described in Example 4.
• the precoat method was used for H-271, and the cocktail method was used for CH-271 and CH-296.
• Plates were washed with RPMI 640 medium containing 0% FCS, 50 units "ml penicillin and 50 zgZml streptomycin. Subsequently, prepared from the pre-stimulated mouse spleen cells prepared according to the method described in Example 6. CD 3 positive T cells were subjected to viral infection for 3 hours with 10 5 Ko ⁇ Ka ⁇ per Ueru. Thereafter, 10% FCS, 500 units of recombinant mouse interleukin one 1 alpha, 10 ng / ml of pairs RPMI 1640 medium containing the recombinant mouse interleukin-12, 50 units Zml of penicillin and 50 / gZml of streptomycin was added, and the culture was continued for another 48 hours.
• the cells were collected, washed, and the cells were labeled with phycoerythrin (PE, Pharmingen) -labeled anti-mouse CD4 monoclonal antibody (Pharmingen) and propionium iodide (P
• the cells were subjected to flow cytometry (excitation wavelength: 488 nm, emission wavelength: 515-545 nm and 562-588 nm) using FACS Vantage to detect live cells.
• the two-dimensional analysis of CD4 antigen expression and EGFP expression was performed to calculate the gene transfer efficiency into each of CD4 positive cells and CD4 negative cells, and the results are shown in Table 12 and Table 12. Is the result of four experiments.
• Table 1 2 The two-dimensional analysis of CD4 antigen expression and EGFP expression was performed to calculate the gene transfer efficiency into each of CD4 positive cells and CD4 negative cells, and the results are shown in Table 12 and Table 12. Is the result of four experiments.
• Table 1 2 Table 1 2
• the CD44 antigen expresses 98% or more of both CD4 positive cells and CD4 negative cells, and when retrovirus infection similar to the above was performed using an anti-CD44 monoclonal antibody, It was thought that the gene transfer efficiency was increased independently of the expression of the CD4 antigen in the cells, but the results in Table 12 support this.
• Example 10 The experimental results of Examples 8 and 9 described above have a very significant meaning.
• the incubator is coated with a mixture (cocktail) of a target cell-specific antibody and a virus-binding functional substance, and the incubator is placed on the incubator.
• a mixture cocktail
• the incubator is placed on the incubator.
• Monoclonal antibodies against 80 ⁇ g / m 1 CH-271 and various cell surface antigens (anti-CD4, anti_CD8, anti-CD44, anti-CD49c, anti-CD49d and anti-CD4) CD49e antibody; all manufactured by Pharmingen) 1 ⁇ g / m 1 was used, and a 24-well microphone-mouth plate for untreated cell culture was coated by the cocktail method according to the method described in Example 4. .
• Target cells include K562 (human chronic myeloid leukemia cells, ATCC CCL-243), HSB-2 (human acute lymphocytic leukemia cells, CCRF-HSB-2, ATCC CCL -1 20.1), MOLT_3 (human acute lymphocytic leukemia cells, ATCC CRL-155 2), TF-1 (human erythroleukemia cells, ATCC CRL-203) was. These cells were subjected to FACS analysis using the above-mentioned various monoclonal antibodies, and the expression rate of the antigen corresponding to the antibodies was measured.
• Table 14 shows the results. These results are the average of the results of three experiments. Table 14
• the gene transfer efficiency is determined by the gene transfer efficiency of each cell without the addition of antibody.
• the positive rate (%) in the FACS measurement was shown as follows, respectively.
• CH-271 as a functional molecule with retrovirus binding ability
• the gene transfer efficiency is determined by the gene transfer efficiency of each cell without the addition of antibody.
• the positive rate (%) in the FACS measurement was shown as follows, respectively.
• Human myeloid leukemia cells HL-60 (ATCC CCL-240) cultured in RPMI 1640 medium containing 10% FCS, 5 ° units Zml of penicillin and 50 / igZml of streptomycin, From the day before the infection experiment, the cells were transferred to the above-mentioned medium containing various concentrations of deferoxamin (manufactured by Sigma), and pre-cultured at 37 ° C for 20 hours in the presence of 5% carbon dioxide. After the cells washed with fresh medium without Defuerokisa Min in use, and used in the following infection experiments were prepared such that 2 X 1 0 5 cells ZML.
• the supernatant of the TKNeovirus prepared in Example 2 was adjusted to 32.5 cfuZml in DMEM, the culture supernatant of NIH / 3T3 cells (ATCC CRL-1658), and the culture supernatant of CRIP cells. It was diluted and used for the following operation. Add 0.5 ml of 32 ⁇ g / ml CII-296 per well to a 24-well microplate for untreated surface cell culture, leave at room temperature for 2 hours, and then in 30% with 2% BSA for 30 minutes Blocked and washed with PBS.
• the plates per Ueru virus solution 1 m 1 above and 2 X 1 0 4 pieces of NIH / 3 T 3 cells were added and 3 7 ° C De ⁇ incubation. Thereafter, the cells were cultured in a selective medium containing 0.75 mgZml of G418 for 10 days, and the number of colonies that appeared was counted.
• the ratio of the number of G418-resistant Knees to the number of Knees obtained in a medium not containing G418 was defined as the gene transfer efficiency. The results are shown in Table 18.
• the transfection efficiency was shown as a relative efficiency (%) to the control.
• the NIH was higher than when the virus was diluted with DMEM.
• NIH / 3T3 cells are the parent strain of many packaging cells, such as ⁇ > CRIP cells and GP + EmVAml2 cells used for preparing the TKNeo virus vector-producing cells used in this experiment.
• the activity of inhibiting retrovirus infection was found in the culture supernatant of the cells, indicating that the inhibitor was also contained in the virus supernatant prepared using similar packaging cells. Suggests.
• Example 12 To remove the virus infection inhibitor shown in Example 12 Method was used.
• the TKNeovirus supernatant prepared in Example 2 was diluted with the culture supernatant of CR IP cells to 500 OcfuZml, and further diluted 2-fold with DMEM. used.
• Recombinant retrovirus-producing cells obtained by introducing retroviral vector-plasmid pLEIN into ⁇ > CRIP cells were cultured in DMEM containing 10% CS.
• the medium When grown to semi-confluence on a 1 Ocm ⁇ plate, the medium contains 7 ml of RPMI 1640 containing 10% FCS, or 5 mM sodium butyrate (Nacalai Tesque) and 10% FCS. 7ml RPM I 1 6
• the supernatant was filtered through a 0.45 micron filter to obtain a virus supernatant.
• the titer of the virus supernatant was measured by the method described in Example 2.
• the titer of the virus solution without sodium butyrate was 3.3 ⁇ 10 4 cfu / mU.
• the titer of the virus solution containing 5 mM sodium butyrate was 2 ⁇ 10 6 cfu / ml.
• Sodium butyrate has the effect of arresting the cell cycle, suppressing cell proliferation, and inducing differentiation, and may have adverse effects on infected cells. The following method was used to remove sodium butyrate contained in the virus solution.
• HL-60 cells were used as target cells.
• a plate coated with CH-296 by the method described in Example 12 0.5 ml of the above virus solution was added per 1 ⁇ l and incubated at 37 ° C. for 3 hours to remove virus particles. Contacted and held on CH-296. After the incubation, the plate was washed three times with PBS, and then 0.5 ml of RPMII 640 medium containing 10% FCS containing 5 ⁇ 10 4 HL-60 cells was added. As a control, a suspension of 5 ⁇ 10 4 HL-60 cells in 0.5 ml of the above virus solution was immediately added to a plate coated with CH-296.
• the gene transfer efficiency in the control experiment was higher in the virus solution prepared by adding sodium butyrate, confirming the effect of sodium butyrate in virus preparation.
• growth in the supernatant using sodium butyrate Although the number of cells was not used, it was 1 to 4 or less, and it was confirmed that the growth of cells was inhibited by sodium butyrate.
• virus particles were brought into contact with and retained on the plate coated with CH-296 in advance, the cell growth inhibition observed in the control experiment and the supernatant using sodium butyrate was not observed.
• DEAE-dextran (manufactured by Sigma) was dissolved in PBS so as to have a concentration of 1 OmgZml, then sterile-filtered with a 0.22 micron filter, and provided for plate coating.
• a 1-ml mixture of 10 volumes of PBS and 1 volume of the above DEAE-dextran solution was added per 1 ⁇ l of a surface-treated 6-well plate for cell culture (Iwaki Glass Co., Ltd.). ⁇ Incubated. After removing the DEAE-dextran solution from the plate, adding 2 ml of 8% 8 solution at 2 ml / well and treating for 30 minutes, the plate was washed three times with 2 ml of PBS / well. As a control, a plate was prepared by performing the same operation using PBS instead of the DEAE-dextran solution.
• virus supernatant was prepared by the above-described method of adding sodium butyrate. This dilution virus solution diluted in DMEM 20 ml virus supernatant 1 volumes of having containing a 10% CS to (1. 6 X 10 6 cfuZ m l) was added in 1 Ueru per lml, 37. Incubated at C for 2 hours.
• the gene transfer efficiency was represented by the ratio (%) of EGFP positive cells to all cells. As shown in Table 20, DEAE-dextran also has a retrovirus binding activity, indicating that it can be used in the gene transfer method of the present invention.
• Example 15
• a centrifuge tube (50 ml, 1 volume polypropylene conical tube, manufactured by Falcon) used for infecting cells with the retrovirus was coated with CH-296 according to the following procedure. That is, 3 ml of PBS containing 40 / igZml of CH—296 was gently placed in the bottom of a centrifuge tube, and incubated at 4 ° C. for 16 hours in an upright state. Next, the CH—296 solution was replaced with 3.5 ml of PBS containing 2% BSA and incubated for 30 minutes at room temperature. After that, 5 ml of Hanks' balanced salt solution (HBSS, manufactured by Gibco ) To wash the centrifuge tube.
• HBSS Hanks' balanced salt solution
• the binding of DOL retrovirus to the bottom of the centrifuge tube of CH-296 is as follows. I went. That is, put 5 ml each of the above DOL virus supernatant stock solution, 10-fold dilution solution, or 100-fold dilution solution into a centrifuge tube, and centrifuge at 2900 Xg, 25 ° C, for 3 hours to forcibly Retrovirus was conjugated on CH-296.
• NI HZ3 T3 cells Gene transfer into NI HZ3 T3 cells was performed using a CH-296 coated centrifuge tube to which the retrovirus was forcibly bound by centrifugation. That is, 1 ⁇ 10 5 NI HZ3T3 cells were placed in the above-mentioned CH-296 coated centrifuge tube in which the virus solution diluted in each step was centrifuged, and incubated at 37 ° C. for 3 hours (hereinafter referred to as centrifugation). ). The microplate was also incubated under the same conditions (hereinafter, binding method). As a control, a microplate coated with CH-296 was mixed with a virus solution and a NIH / 3T3 cell mixture, and incubated at 37 ° C for 3 hours.
• Fig. 4 shows the results.
• the horizontal axis shows the dilution rate of the virus supernatant
• the vertical axis shows the gene transfer efficiency.
• the white outline shows the results obtained by the supernatant method
• the shaded area shows the results obtained by the binding method
• the black area shows the results obtained by the centrifugation method.
• Table 21 shows the results of titer measurement of the virus supernatant collected after the virus binding operation under the above-mentioned centrifugation and in a stationary state.
• the recovered supernatant has a titer of about 80 to 90% before the operation, whereas when the supernatant is forcibly bound under centrifugation, The refining system had a titer of less than 10% before the operation. This indicates that more virus particles were bound on CH-296 by centrifugal force. Also, since the amount of virus contained in PBS after washing the centrifuge tube after centrifugation is about 2% of the original solution, and whether or not this washing operation has little effect on gene transfer efficiency, When used, it was suggested that the virus particles were firmly retained on CH-296.
• the virus supernatant prepared using the GP + E86 cells described in Example 14 was diluted to 1 ⁇ 10 5 cfu / ml with a culture supernatant of NIH / 3T3 cells.
• NIH / 3T by centrifugation and the method of infecting virus by sedimentation on cells by centrifugal force (centrifugal infection, see WO 95 10619)
• the efficiency of gene transfer to three cells was compared. That is, the above-mentioned virus solution was added to a centrifuge tube coated with CH-296, and the mixture was centrifuged at 30 ° C for 2 hours at 2900 X g, and then the centrifuge tube was washed with PBS. C, infected for 4 hours (centrifugation method), add cells to a centrifuge tube coated with CH-296, incubate for 2 hours, add virus solution, and centrifuge at 2900 X g, 4 hours at 30 ° C The transfection was performed for each of the transfected cells (centrifugal infection method).
• the centrifugal tube was coated with CH-296 by the method described above, and 1 ⁇ 10 5 NIH / 3T3 cells were used for each gene transfer operation. 6 after infection After reseeding on an O mm plate and culturing for 2 days, the transfection efficiency of the EGFP gene was examined by the flow cytometry method described in Example 4. Fig. 5 shows the results.
• the efficiency of gene transfer by the centrifugation method was higher than that by the centrifugal infection method. This is probably because the infection inhibitor present in the virus solution was removed by the washing operation.

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